Inflammasome and Caspase-1 Activity Characterization and Evaluation: An Imaging Flow Cytometer–Based Detection and Assessment of Inflammasome Specks and Caspase-1 Activation

Nagar, Abhinit; DeMarco, Richard; Harton, Jonathan A.

doi:10.4049/jimmunol.1800973

Cited by 26 publications

(31 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…First, IL-1β is stored as an inactive a precursor (pro-IL-1β) and is cleaved into its mature form by caspase-1 via inflammasome activation so transcriptional upregulation is not a proxy for functional IL-1 release [22]. Second, IL-1β is released by inflammasome-mediated pore formation and rapid inflammatory cell death making these cells difficult to identify in situ [23]. To circumvent challenges and identify which cell populations predicted to release IL-1β we applied the bleomycin-induced fibrosis model in mice expressing a transgene for apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) tagged with the fluorescent protein mCitrine (Figure 3A).…”

Section: Resultsmentioning

confidence: 99%

Myeloid-mediated IL-1R signaling in immuno-responsive Thy-1 negative fibroblasts is critical for pulmonary fibrosis

Abebayehu

et al. 2021

Preprint

View full text Add to dashboard Cite

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with poorly defined pathogenic mechanism and no cure. It is characterized by chronic inflammation, myofibroblast accumulation, and aberrant extracellular matrix (ECM) remodeling. Fibrosis progression is considered to occur due to sustained aberrant fibroblast mechanotransduction: sensing "normal" soft tissue as stiff scarred tissue leading to the overproduction of ECM that then stiffens the microenvironment, thus reinforcing a progressive, stiffness-dependent fibrotic program. How chronic inflammation leads to aberrant mechanotransduction is not well understood. Thy-1 is a regulator of mechanotransduction in fibroblasts. Thy-1 expression is lost in fibroblastic foci, the active sites of fibrosis, although the mechanism of this loss is unknown. We demonstrate that in IPF tissue, the αSMA+ fibroproliferative foci express the Type 1 IL-1 receptor (IL-1RI) and IL-1RI-deficient mice did not develop bleomycin-induced pulmonary fibrosis. Using ASC speck formation during inflammasome activation as a marker of mature IL-1β release, we identified the immune compartment as the source of active IL-1β during bleomycin-induced fibrosis. Furthermore, incubating mouse lung fibroblasts on soft (2kPa) hydrogels with IL-1β was sufficient to reduce Thy-1 surface expression and induce αvβ3 integrin activation. As expected, Thy-1 negative fibroblasts exhibited elevated αvβ3 integrin activation but surprisingly, Thy-1 negative fibroblasts also expressed higher levels of IL-1RI, potentially linking the immuno-responsive and mechanosensitivity of this fibroblast subpopulation. Leveraging the non-resolving fibrosis that occurs in Thy-1-/- mice, we observed that crossing Thy-1-/- mice onto the IL-1RI-/- background was sufficient to reduce fibrosis. Together, these data indicate that Thy-1 negative fibroblasts are an immuno-responsive subpopulation that also display altered mechanotransduction, potentially serving as the link between the noted inflammation and aberrant mechanotransduction observed in IPF.

show abstract

Section: Resultsmentioning

confidence: 99%

Myeloid-mediated IL-1R signaling in immuno-responsive Thy-1 negative fibroblasts is critical for pulmonary fibrosis

Abebayehu

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…A stop gate of 10 4 cells was set on the GFP-positive gate. The percentage of cell containing ASC specks was determined by analyzing the height (H), width (W), and area (A) of the GFP pulse area (high H:A and low W:A indicates speck positive cells) as described previously ( 78 ).…”

Section: Methodsmentioning

confidence: 99%

NLRP3 Sensing of Diverse Inflammatory Stimuli Requires Distinct Structural Features

et al. 2020

Self Cite

View full text Add to dashboard Cite

The NLRP3 inflammasome is central to host defense and implicated in various inflammatory diseases and conditions. While the favored paradigm of NLRP3 inflammasome activation stipulates a unifying signal intermediate that de-represses NLRP3, this view has not been tested. Further, structures within NLRP3 required for inflammasome activation are poorly defined. Here we demonstrate that while the NLRP3 LRRs are not auto-repressive and are not required for inflammasome activation by all agonists, distinct sequences within the NLRP3 LRRs positively and negatively modulate inflammasome activation by specific ligands. In addition, elements within the HD1/HD2 "hinge" of NLRP3 and the nucleotide-binding domain have contrasting functions depending upon the specific agonists. Further, while NLRP3 1-432 is minimally sufficient for inflammasome activation by all agonists tested, the pyrin, and linker domains (1-134) function cooperatively and are sufficient for inflammasome activation by certain agonists. Conserved cysteines 8 and 108 appear important for inflammasome activation by sterile, but not infectious insults. Our results define common and agonist-specific regions of NLRP3 that likely mediate ligand-specific responses, discount the hypothesis that NLRP3 inflammasome activation has a unified mechanism, and implicate NLRP3 as an integrator of agonist-specific, inflammasome activating signals.

show abstract

“…Development of tools to monitor inflammasome signaling within single cells is still needed. Some tools have been developed (Nagar et al, 2019;Tzeng et al, 2016). Once these tools (and others) become more widespread in use, a detailed analysis of the mechanisms and consequences of inflammasome activity can be explored.…”

Section: Immunitymentioning

confidence: 99%

Inflammasomes: Threat-Assessment Organelles of the Innate Immune System

2019

View full text Add to dashboard Cite

Inflammasome and Caspase-1 Activity Characterization and Evaluation: An Imaging Flow Cytometer–Based Detection and Assessment of Inflammasome Specks and Caspase-1 Activation

Cited by 26 publications

References 45 publications

Myeloid-mediated IL-1R signaling in immuno-responsive Thy-1 negative fibroblasts is critical for pulmonary fibrosis

Myeloid-mediated IL-1R signaling in immuno-responsive Thy-1 negative fibroblasts is critical for pulmonary fibrosis

NLRP3 Sensing of Diverse Inflammatory Stimuli Requires Distinct Structural Features

Inflammasomes: Threat-Assessment Organelles of the Innate Immune System

Contact Info

Product

Resources

About